U.S. patent number 11,385,179 [Application Number 16/500,869] was granted by the patent office on 2022-07-12 for target molecule density determination in a fluorescence image.
This patent grant is currently assigned to HOFFMANN-LA ROCHE, INC.. The grantee listed for this patent is HOFFMANN-LA ROCHE INC.. Invention is credited to Eldad Klaiman.
United States Patent |
11,385,179 |
Klaiman |
July 12, 2022 |
Target molecule density determination in a fluorescence image
Abstract
The method includes receiving a digital image of a slide, the
slide including the tissue sample, a target dot and a fluoro dot.
The digital image includes intensity values of the tissue sample,
the target dot and the fluoro dot. The method further includes
receiving pixel intensities of the fluoro dot depicted in the
image; pixel intensities of the target dot depicted in the image;
pixel intensities of an area of the tissue sample depicted in the
image; fluorescence-inducing molecule density information
indicative of the known density of the fluorescence-inducing
molecules in the fluoro dot; target molecule density information
indicative of the known density of the target molecules in the
target dot; and computing the density of target molecules in the
area of the tissue sample as a function of the received pixel
intensities and the received molecule density information.
Inventors: |
Klaiman; Eldad (Penzberg,
DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
HOFFMANN-LA ROCHE INC. |
Little Falls |
NJ |
US |
|
|
Assignee: |
HOFFMANN-LA ROCHE, INC. (Little
Falls, NJ)
|
Family
ID: |
1000006424378 |
Appl.
No.: |
16/500,869 |
Filed: |
April 13, 2018 |
PCT
Filed: |
April 13, 2018 |
PCT No.: |
PCT/EP2018/059528 |
371(c)(1),(2),(4) Date: |
October 04, 2019 |
PCT
Pub. No.: |
WO2018/189370 |
PCT
Pub. Date: |
October 18, 2018 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20200033267 A1 |
Jan 30, 2020 |
|
Foreign Application Priority Data
|
|
|
|
|
Apr 13, 2017 [EP] |
|
|
17166661 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G06T
7/0012 (20130101); G01N 21/6458 (20130101); G01N
33/5005 (20130101); G01N 21/6486 (20130101) |
Current International
Class: |
G06K
9/00 (20220101); G01N 33/50 (20060101); G01N
21/64 (20060101); G06T 7/00 (20170101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1234114 |
|
Nov 1999 |
|
CN |
|
101688838 |
|
Mar 2010 |
|
CN |
|
103620413 |
|
Mar 2014 |
|
CN |
|
104634962 |
|
May 2015 |
|
CN |
|
1774292 |
|
Apr 2016 |
|
EP |
|
WO-00/51058 |
|
Aug 2000 |
|
WO |
|
WO-01/35074 |
|
May 2001 |
|
WO |
|
WO-01/59503 |
|
Aug 2001 |
|
WO |
|
Other References
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2019, issued in corresponding PCT Application No.
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Written Opinion of the International Searching Authority
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filed Apr. 13, 2018. cited by applicant .
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construction of protein microarrays" . 2003, Analytical
Biochemistry, vol. 312, p. 113-124, Academic Press. cited by
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WILEY-VCH Verlag GmbH & Co, KGaA, Weinheirn. cited by applicant
.
Roth et al., "Enzyme-Based Fluorescence Amplification for
Immunohistochemistry and in Situ Hybridization," 2005, Molecular
Morphology in Human Tissues, CRC Press. cited by applicant .
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Immunological Methods, vol. 150, p. 145-149, Etsevier Science
Publishers B.V. cited by applicant .
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Signals in Histochemical Stains," 1992, The Journal of
Histochemistry and Cytochemistry, vol, 40, No. 10, p. 1457-1463,
The Histochernical Society, Inc. cited by applicant .
Shindler, Double Immunofluorescent Staining Using Two Unconjugated
Primary Antisera Raised in the Same Species, 1996, The Journal of
Histochemistry and Cytochemistry, vol. 44, No. 11, p. 1331-1335,
The Histochemical Society, Inc. cited by applicant .
Office Action for Corresponding Japanese Patent Application No.
2019-553845 dated Feb. 8, 2022 and English Translation thereof.
cited by applicant .
Office Action for Corresponding Chinese Patent Application No.
201880024609.9 dated Dec. 30, 2021 and English Translation thereof.
cited by applicant.
|
Primary Examiner: Yang; Wei Wen
Attorney, Agent or Firm: Harness, Dickey & Pierce,
P.L.C.
Claims
The invention claimed is:
1. An image analysis method for determining the density of target
molecules in a tissue sample depicted in a fluorescence image, the
method comprising: receiving, by an image analysis system, a
digital image of a slide, the slide comprising the tissue sample, a
target dot and a fluoro dot, the fluoro dot comprising a known
density of fluorescence-inducing molecules, the target dot
comprising a known density of target molecules, the tissue sample
and the target dot having been stained in the same staining
procedure with the same type of fluorescence-inducing molecules as
contained in the fluoro dot, the digital image comprising intensity
values of the tissue sample, intensity values of the target dot and
intensity values of the fluoro dot, the fluorescence-inducing
molecules being adapted to directly or indirectly bind to the
target molecules during the staining procedure; receiving, by the
image analysis system: pixel intensities of the fluoro dot depicted
in the image; pixel intensities of the target dot depicted in the
image; pixel intensities of an area of the tissue sample depicted
in the image; fluorescence-inducing molecule density information
indicative of the known density of the fluorescence-inducing
molecules in the fluoro dot; and target molecule density
information indicative of the known density of the target molecules
in the target dot; and computing the density of target molecules in
the area of the tissue sample as a function of the pixel
intensities of the fluoro dot, the target dot, the area of the
tissue sample, the fluorescence-inducing molecule density
information and the target molecule density information.
2. The image analysis method of claim 1, the computation of the
density of the target molecules in the tissue area comprising:
computing the average intensity I.sub.FID_AVG of the pixel
intensities of the fluoro dot according to:
.times..times..times..times..times..times..times..times.
##EQU00005## computing the average intensity I.sub.TaD_AvG of the
pixel intensities of the target dot according to:
.times..times..times..times..times..times. ##EQU00006## computing
the ratio R of fluorescence-inducing molecules per target molecule
in the target dot according to: .times..times..times..times.
##EQU00007## computing the density .delta..sub.Target_in_Tissue of
the target molecules in the area of the tissue sample according to:
.delta..times..times..times..times..times..times..times..times..times..ti-
mes..times..times..times..times..times..times..times..times..times..times.-
.times..delta..times..times..times. ##EQU00008## wherein
.delta..sub.Target_In_Target_dot is the density of target molecules
in the target dot.
3. The image analysis method of claim 1, further comprising:
coating an area of the slide with the target molecules, thereby
creating the target dot.
4. The image analysis method of claim 3, the coating of the area of
the slide being performed using protein microarray technology.
5. The image analysis method of claim 1, further comprising
generating the target dot, the generation comprising: attaching
reference cells homogeneously to an area of the slide, thereby
generating the target dot, the reference cells expressing or
comprising a known number of the target molecules.
6. The image analysis method of claim 5, the method further
comprising, before the attaching of the reference cells is
performed: Before the attaching of the reference cells is
performed, experimentally determining the average number of the
target molecules which are expressed in or are contained in a set
of reference cells; and counting the number of cells attached to
the target dot.
7. The image analysis method of claim 6, the experimental
determination comprising: Processing a reference tissue sample, in
particular a FFPE tissue sample, for transforming the reference
tissue sample into a homogeneous suspension of reference cells;
Experimentally determining the average number of target molecules
comprised in the reference cells in the suspension or in a sub-set
of the reference cells in the suspension.
8. The image analysis method of claim 1, the experimental
determination comprising performing a mass spectroscopy analysis or
performing a flow cytometry analysis for a quantitative
determination of target molecules in the reference cells.
9. The image analysis method of claim 1, wherein the ratio of
fluorescence-inducing molecules that bind to a single target
molecule during the staining procedure is unknown and depends on
one or more parameters of the staining protocol.
10. The image analysis method of claim 1, wherein the staining
protocol is a tyramide signal amplification staining protocol.
11. The image analysis method of claim 1, the binding of a
fluorescence-inducing molecule to a single target molecule being
selected from a group comprising: a covalent or ionic binding to
the target molecule or to one or more intermediary molecules
connected with the target molecule; dipole-dipole interactions,
Van-der-Waals interactions or hydrogen binding to the target
molecule or one or more intermediary molecules connected with the
target molecule; an antigen-antibody binding; a hybridization of
nucleic acid sequences.
12. The image analysis method of claim 1, the fluorescence-inducing
molecules being fluorophores or enzymes that trigger the emission
of fluorescence signals by other molecules in spatial proximity to
the fluorescence-inducing molecules.
13. The image analysis method of claim 1, the fluoro dot comprising
a known density of further fluorescence-inducing molecules of at
least one further type of fluorescence-inducing molecules, the
tissue sample and the target dot both having been stained in
addition with the further fluorescence-inducing molecules, the
method comprising: Receiving a further digital image of the slide,
the further digital image comprising intensity values of the tissue
sample, intensity values of the target dot and intensity values of
the fluoro dot, the intensity values of the fluoro dot, the target
dot and the tissue sample correlating with the number of the
further fluorescence-inducing molecules in the fluoro dot, the
target dot and the tissue dot, the further fluorescence-inducing
molecules being adapted to directly or indirectly bind to the
target molecules during the staining procedure; receiving: further
pixel intensities of the fluoro dot depicted in the further image;
further pixel intensities of the target dot depicted in the further
image; further pixel intensities of the area of the tissue sample
depicted in the further image; further fluorescence-inducing
molecule density information indicative of the known density of the
further fluorescence-inducing molecules in the fluoro dot;
computing a further density of target molecules in the area of the
tissue sample as a function of the further pixel intensities of the
fluoro dot, the target dot, the area of the tissue sample, the
further density information of the further fluorescence-inducing
molecules and the target molecule density information.
14. The image analysis method of claim 1, the target molecule being
selected from a group comprising: a biomarker molecule; a primary
antibody capable of selectively binding to the biomarker molecule
in a predefined molecular ratio; a secondary antibody capable of
selectively binding to the primary antibody in a predefined
molecular ratio.
15. An image analysis system configured for determining the density
of target molecules in a tissue sample depicted in a fluorescence
image, the system comprising a storage medium and one or more
processors configured for: receiving a digital image of a slide,
the slide comprising the tissue sample, a target dot and a fluoro
dot, the fluoro dot comprising a known density of
fluorescence-inducing molecules, the target dot comprising a known
density of target molecules, the tissue sample and the target dot
having been stained in the same staining procedure with the same
type of fluorescence-inducing molecules as contained in the fluoro
dot, the digital image comprising intensity values of the tissue
sample, intensity values of the target dot and intensity values of
the fluoro dot, the fluorescence-inducing molecules being adapted
to directly or indirectly bind to the target molecules during the
staining procedure; receiving: pixel intensities of the fluoro dot
depicted in the image; pixel intensities of the target dot depicted
in the image; pixel intensities of an area of the tissue sample
depicted in the image; and fluorescence-inducing molecule density
information indicative of the known density of the
fluorescence-inducing molecules in the fluoro dot; target molecule
density information indicative of the known density of the target
molecules in the target dot; and computing the density of target
molecules in the area of the tissue sample as a function of the
received pixel intensities of the fluoro dot, the target dot, the
area of the tissue sample, the fluorescence-inducing molecule
density information and the target molecule density
information.
16. A slide comprising a tissue sample, a target dot and a fluoro
dot, the fluoro dot comprising a known density of
fluorescence-inducing molecules, the target dot comprising a known
density of target molecules.
17. The slide of claim 16, the slide with the tissue sample and the
target dot having been stained in the same staining procedure with
the same type of fluorescence-inducing molecule as contained in the
fluoro dot, the fluorescence-inducing molecules being adapted to
directly or indirectly bind to the target molecules during the
staining procedure.
18. The slide of claim 16, the fluoro dot of the slide comprising a
known density of further fluorescence-inducing molecules of at
least one further type of fluorescence-inducing molecules, the
further fluorescence-inducing molecules being adapted to directly
or indirectly bind to the target molecules during the staining
procedure.
19. The slide of claim 16, the target dot being an area of the
slide that is coated with a known number of the target molecules or
that is coated with a known number of cells, the cells expressing
or comprising a known number of the target molecules.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national phase under 35 U.S.C. .sctn. 371 of
PCT International Application No. PCT/EP2018/059528 which has an
International filing date of Apr. 13, 2018, which claims priority
to European patent application number EP 17166661.3 filed Apr. 13,
2017.
FIELD OF THE INVENTION
The invention relates to the field of image analysis, and more
particularly to the field of fluorescence image-based
quantification of target molecules.
BACKGROUND AND RELATED ART
Fluorescence microscopes are commonly used for studying particular
cells which have been labeled with fluorescence-labeled antibodies
or other fluorescence labeled proteins in order to detect the
occurrence and, at least roughly, the quantity of a particular
molecule of interest. For example, fluorescence microscope images
are often generated and analyzed in order to determine if and to
what amount a particular biomarker (being an indicator of a
physiological state, e.g. a disease, in particular a cancer or
cancer sub-type) is present in a cell.
Typically, fluorescence intensity and biomarker expression are
positively correlated and the intensity of a fluorescence signal is
used as an indicator for the amount of the biomarker.
However, due to various instrumental factors, the same sample
imaged on two microscopes or even on the same microscope at
different times may produce highly divergent readings. Second, the
ratio of antibody molecules which bind to the biomarker and/or the
ratio of fluorescent dye molecules that bind to an antibody
molecule may strongly depend on process parameters of the staining
process (such as temperature, incubation time, buffer, type of
fluorophore molecule, etc). Therefore, the fluorescence intensity
signal in fluorescence images does typically not allow an accurate
determination of the number of biomarker molecules expressed in a
cell and thus does not allow to accurately compare expression
levels of two tissue samples having been stained in different
staining procedures by means of fluorescence microscopy.
U.S. Pat. No. 9,395,283 B1 describes the generation of homogeneous
cell blocks (i.e. FFPE and non-FFPE) for using sections of the cell
block as a positive control for tissue based biomarker studies. The
FFPE cell section has a defined number of cells with defined ratio.
Said cell blocks are used as a standard for sensitivity and
specificity evaluation in histological assay, but they are not used
for calibrating fluorescence signals for quantification target
molecules in a tissue sample.
European patent EP 1774292 E1 describes a calibration slide for
fluorescence detection instruments and a process of preparing the
same.
US 2004/0060987 A1 describes a method for detecting analytes using
arrays of binding moieties. The arrays are attached to glass
slides. Fluorescent signals obtained from the slides are analyzed
by a digital image subtraction method. The glass slides can
comprise calibration spots with known amounts of a FluoSphere.
SUMMARY
It is an objective of the present invention to provide for an
improved image analysis method and image analysis system for
determining the density of target molecules in a tissue sample
depicted in a fluorescence image as specified in the independent
claims. Embodiments of the invention are given in the dependent
claims. Embodiments of the present invention can be freely combined
with each other if they are not mutually exclusive.
In one aspect, the invention relates to an image analysis method
for determining the density of target molecules in a tissue sample
depicted in a fluorescence image. The method comprises: receiving,
by an image analysis system, a digital image of a slide; the slide
comprises the tissue sample, a target dot and a fluoro dot; the
fluoro dot comprises a known density of fluorescence-inducing
molecules; the target dot comprises a known density of target
molecules; the tissue sample and the target dot have been stained
in the same staining procedure with the same type of
fluorescence-inducing molecules as contained in the fluoro dot; the
digital image comprises intensity values of the tissue sample,
intensity values of the target dot and intensity values of the
fluoro dot; the fluorescence-inducing molecules are adapted to
directly or indirectly bind to the target molecules during the
staining procedure; receiving, by the image analysis system: pixel
intensities of the fluoro dot depicted in the image; pixel
intensities of the target dot depicted in the image; pixel
intensities of an area of the tissue sample depicted in the image;
fluorescence-inducing molecule density information indicative of
the known density of the fluorescence-inducing molecules in the
fluoro dot; target molecule density information indicative of the
known density of the target molecules in the target dot; computing,
by the image analysis system, the density of target molecules in
the area of the tissue sample as a function of the pixel
intensities of the fluoro dot, the target dot, the area of the
tissue sample, the fluorescence-inducing molecule density
information and the target molecule density information.
Embodiments of the invention may have the advantage that--by using
a new form of slide that comprises a fluoro dot and a target dot as
described above and by using density information of the
fluorescence-inducing molecules or target molecules in the
respective dots--it is now possible to calibrate the intensity
information of a fluorescence image in a way that the absolute
number of target molecules in a tissue sample depicted in a
fluorescence image can accurately and exactly be determined fully
automatically by means of an image analysis method.
In one aspect, the method allows to normalize acquired intensity
information by abstracting away the impact of optical components of
the image acquisition system, e.g. the loss of intensity that
occurs when fluorescence light traverses lenses and other
components of a microscope and which may result in an
underestimation of the number of target molecules.
In a further beneficial aspect, the method allows to normalize
acquired intensity information by abstracting away the impact of a
bad quality of the light source that stimulates the fluorophore to
emit fluorescent light. For example, in case the light source of a
first microscope is stronger than the light source of a second
microscope, the fluorescence signal obtained by the first
microscope for a particular slide and a particular tissue sample
will be stronger than the fluorescence signal obtained by the
second microscope for the same slide. However, as these factors
affect the fluorescence-inducing molecules in the fluoro dot in the
same way as the fluorescence-inducing molecules in the tissue
sample, this effect can be computationally eliminated.
In a further beneficial aspect, the method allows to
computationally eliminate the impact of parameters of the staining
protocol on the fluorescence intensity. For example, fluorescence
microscopy may be used for determining whether a particular cancer
patient benefits from a particular treatment scheme. To test the
efficiency of the anti-cancer drug, one or more tumor markers of a
first biopsy sample of the patient may be labeled with a
fluorescent stain before treatment for generating a first
fluorescence image. Then, after some weeks of treatment, the same
one or more tumor markers of a second biopsy sample of the patient
may be labeled with the same fluorescent stain and are used for
generating a second fluorescence image. As the number of
fluorophore molecules that bind to a particular tumor marker (i.e.,
a form of target protein) may depend on the temperature and
duration of the staining step, on the chemical composition of the
staining buffer and other factors, it is currently not possible to
accurately quantify the effect of a particular anti-cancer drug on
the expression level of a tumor marker, because other factors
(related to the sample handling and preparation procedures, the
staining protocol, the hardware of the microscope, etc.) have also
a significant impact on the intensity of a fluorescence signal.
However, by using two "calibration dots", i.e., the target dot and
the fluoro dot, and the respectively known molecule densities, it
may be possible to computationally eliminate the effect of said
error sources on the fluorescence intensity, and to computationally
determine the "real" number of target molecules contained in a
particular tissue sample on the slide that comprises also the
target dot and the fluoro dot. That the two calibration dots and
the actually analyzed tissue sample are contained on the same slide
may have the benefit that the molecules in the two dots and in the
sample were subjected to the same staining procedure and that the
pixel intensities received for the tissue sample region of the
slide were received by the same image acquisition hardware as the
pixel intensities of the calibration dots.
Embodiments of the invention combine knowledge derived from
different domains such as image processing, microscopy, in
particular fluorescence imaging, and wet lab procedures for
enabling a more accurate assessment of the amount of target
molecules contained in a sample.
According to embodiments, the computation of the density of the
target molecules in the tissue area comprises: computing the
average intensity I.sub.FID_AVG of the pixel intensities of the
fluoro dot according to
.times..times..times..times..times..times..times..times.
##EQU00001## computing the average intensity I.sub.TaD_AVG of the
pixel intensities of the target dot according to:
.times..times..times..times..times..times. ##EQU00002## computing
the ratio R of fluorescence-inducing molecules per target molecule
in the target dot according to:
.times..times. ##EQU00003## computing the density
.delta.Target_In_Tissue of the target molecules in the area of the
tissue sample according to:
.delta..times..times..times..times..times..times..times..times..times..ti-
mes..times..times..times..times..times..times..times..times..times..times.-
.times..times..times..delta..times..times..times. ##EQU00004##
wherein .delta.Target_In_Target_dot is the density of target
molecules in the target dot.
For example, the average intensities I.sub.FID_AVG and/or
I.sub.TaD_AVG can be computed as the arithmetic mean or as the
median of the pixel intensity values received for the respective
dots.
According to embodiments, the method further comprises coating an
area of the slide with the target molecules, thereby creating the
target dot.
For example, the coating can be performed by coating an area of the
slide with a predefined number of the target molecules, thereby
creating the target dot. The predefined number is then stored. For
example, coating procedure may be performed such that it is ensured
that a defined number of molecules per mm.sup.2 are attached to the
slide. The known molecule density can be printed or written onto
the slide and/or can be stored in digital form in a database in
association with an identifier of the slide or in association with
an identifier of a set of slides coated in the same coating
procedure or according to the same coating protocol. The molecule
that is written or printed onto the slide may also be entered by a
user, e.g. a lab worker, into the database. By using a coating
technique with a known resulting molecule density, also the density
of the target molecules in the target dot is "known", i.e., is
available to the image analysis system, e.g. in the form of a data
value stored in a digital storage medium.
Alternatively, the coating can be performed by coating an area of
the slide with an unknown number of the target molecules, thereby
creating the target dot, and then measuring the density of the
target molecules in the target dot. For example, a particular
single coating procedure may be performed for a large number of
slides, e.g. several 100 or several 1000 or even several 10.000
slides. Then, the target molecule density in the target dot of one
of sad slides or a small sub-set of said slides is empirically
analyzed, e.g. by means of mass spectrometry, for determining the
actual target molecule density in said few analyzed slides. In case
the target molecule is a particular DNA or RNA sequence, the target
molecule density in the target dot can be determined by means of
quantitative PCR. The obtained target molecule density is then
printed or written onto the slide and/or is stored in digital form
in a database in association with an identifier of the slide or in
association with an identifier of all slides coated in said single
coating procedure as described already for the alternative coating
approach.
According to embodiments, the coating of the area of the slide that
shall constitute the target dot is performed using protein
microarray technology.
According to embodiments, the method further comprises generating
the target dot. The generation of the target dot comprises
attaching reference cells homogenously to an area of the slide,
thereby generating the target dot. The reference cells express or
comprise a known number of the target molecules.
For example, the number of the target molecules in the reference
cells can be determined empirically e.g. by mass spectrometry, cell
cytometry, quantitative PCR (in case the target molecule is a DNA
or RNA sequence of interest), etc. For example, a known number of
reference cells may be attached to the slide an unknown number of
the reference cells can be attached to the slide area that is to
form the target dot and the density of the reference cells having
been successfully attached to the slide is determined later after
the target dot was created.
According to embodiments, the method further comprises, before the
reference cells are attached to the slide: experimentally
determining the average number of the target molecules which are
expressed in or are contained in a set of reference cells (known to
comprise the target molecule); and counting the number of cells
attached to the target dot.
Said method may be beneficial, as it may not be necessary to
extract and purify a particular target molecule, typically a
biomarker, e.g. a particular protein or a particular RNA or DNA
sequence, before it is used for coating the target dot with target
molecules. Rather, it is possible to directly coat the target dot
with reference cells known to comprise (e.g. express) the target
molecules.
The reference cells can be attached to the slide by a coating
technique or by generating homogeneous reference cell blocks as
described e.g. in U.S. Pat. No. 9,395,283 and attaching the
reference cell blocks as the target dots to the slides.
According to embodiments, the experimental determination comprises
processing a reference tissue sample for transforming the reference
tissue sample into a homogeneous suspension of reference cells; and
experimentally determining the average number of target molecules
comprised in the reference cells in the suspension or in a sub-set
of the reference cells in the suspension. In addition, the number
of cells per mm.sup.2 of the target dot is determined empirically.
The target molecule density in the target dot is then computed as a
function of the empirically determined reference cell density in
the target dot and the empirically determined average number of
target molecules contained in each reference cell.
Said approach may be beneficial, because some target proteins
cannot be extracted and purified from cells, or the extraction is
highly complex. By coating an area of the slide with reference
cells known to comprise the target molecules, basically any type of
molecule that is comprised in at least one known type of cells can
be used for generating the target dot. Further, the generation of
the target dot by coating an area of the slide with reference cells
comprising the target dot may more closely represent the
environment provided by the tissue sample of the slide for the
interaction of the fluorescence-inducing molecules with the target
molecules.
According to embodiments, the experimental determination comprises
performing a mass spectroscopy analysis or a flow cytometry
analysis for quantitatively determining the number of target
molecules in the reference cells. This may be beneficial, as said
procedures are well established techniques for quantifying a
particular molecule type in a cell or in any other material
context. Although both methods tend to be complex, a growing number
of semi-automated or automated systems exist which allow an
accurate quantification of e.g. a particular protein in a
particular cell or set of cells. It should be considered that this
approach needs to be done only once for a representative sub-set of
reference cells, and the same cell suspension may be used to create
a large number of tissue slides with a respective target dot. Thus,
performing the quantitative analysis for the reference cells to be
used as the cells of the target dot once allows determining the
"real" number of target molecules in a large number of tissue
samples without respectively performing mass spectroscopy or flow
cytometry analysis for each of said tissue samples individually.
Thus, an accurate method of quantifying a target molecule in a
tissue sample may be provided that does not necessarily require a
complex analytical procedure such as mass spectroscopy or flow
cytometry for each individual tissue sample.
According to embodiments, the ratio of fluorescence-inducing
molecules that bind to a single target molecule during the staining
procedure is unknown and depends on one or more parameters of the
staining protocol. For example, the duration, temperature or
solvent used during the staining process or while preparing a
tissue sample for the staining protocol may have an impact on this
ratio and may be an obstacle in determining the target molecule
density based on the fluorescence signal intensity.
According to embodiments, the staining protocol is a tyramide
signal amplification staining protocol ("TSA assay").
The sensitivity of immunohistochemical (IHC) fluorescence labeling
depends on the properties of the fluorophore used for detection and
on the amount of fluorophore present at the sites of antibody
binding. Tyramide signal amplification (TSA, TSA assay) is a
comparatively new enzymatic amplification procedure that deposits
additional fluorophore molecules at the sites of antibody binding,
thereby lowering IHC antigen detection limits. TSA can be utilized
in single or multilabel IHC applications and can be coupled with
quantum dots.
Traditional enzymatic IHC detection methods have utilized the
ability of horseradish peroxidase (HRP) or alkaline phosphatase
(AP) to convert a chromogenic substrate into a colored reaction
product that precipitates at the site of enzymatic activity (Roth
and Baskin, 2005; Roth and Perry, 2005). The sensitivity of HRP or
AP detection can be improved upon by using "layering" techniques
such as peroxidase-anti-peroxidase (PAP) or avidin-biotin complexes
(ABC) that increase the number of enzyme molecules associated with
the primary antibody. However, several factors limit the
sensitivity and utility of these traditional enzymatic
amplification procedures. In particular, multilabeling options and
sensitivity may be limited.
To the contrary, TSA assays (respective kits are available e.g.
from Perkin-Elmer, Waltham, Mass.) are much more sensitive and also
support multilabeling. TSA is based on the ability of HRP, in the
presence of low concentrations of H.sub.2O.sub.2, to convert
labeled tyramide-containing substrate into an oxidized, highly
reactive free radical that can covalently bind to tyrosine residues
at or near the HRP (Bobrow et al., 1992; Adams, 1992; Shindler and
Roth, 1996). Tyramide is prelabeled with a fluorophore, which is
directly visualizable upon its deposition, or a hapten, which is
then detected in subsequent steps with a hapten-specific reagent
linked to a fluorophore or an enzyme molecule that can be used to
deposit chromogen. In contrast to conventional fluorescence IHC
detection methods (which utilize secondary antibodies labeled with
a fluorophore), TSA results in the deposition of many more
fluorescent molecules than can be linked to secondary antibodies.
However, a downside of this amplification is that the degree of
amplification depends on many parameters, e.g. parameters of the
TSA Assay like temperature, staining duration, etc. and thus the
strength of the fluorescence signal of a given number of target
molecules may vary greatly in different TSA assays.
Embodiments of the invention may be particularly useful in the
context of TSA assays, because the signal amplification and
multi-labeling capabilities of the TSA assays can be used without
reducing the accuracy of target molecule density determination,
because the effect of the TSA assay parameters on the fluorescence
signal intensity can be computationally eliminated by taking into
account also the fluorescence intensities of the two calibration
dots and the known molecule densities in the two dots.
According to embodiments, the binding of a fluorescence-inducing
molecule to a single target molecule can be one of the following
options: a covalent or ionic binding to the target molecule or to
one or more intermediary molecules connected with the target
molecule; for example, various covalently as well as non-covalently
bound molecules (primary and/or secondary antibodies, avidine,
biotine, and others) may be used to induce a fluorescence signal
selectively in the vicinity of a target molecule; dipole-dipole
interactions, Van-der-Waals interactions or hydrogen binding to the
target molecule or one or more intermediary molecules connected
with the target molecule; an antigen-antibody binding; for example,
the fluorescent-inducing molecule can covalently bind to a primary
antibody and the primary antibody can selectively bind to the
target protein; according to another example, the
fluorescent-inducing molecule can covalently bind to a secondary
antibody, the secondary antibody can covalently bind to a primary
antibody and the primary antibody can selectively bind to the
target protein; in fact, in case the target dot already comprises
molecule complexes of biomarker molecules and one or more primary
antibodies bound to said biomarker molecule, and in case the number
of said molecule complexes per mm2 target dot as well as the ratio
of biomarker protein to primary antibody (or any other intermediary
molecule) is known, the whole molecule complex constitutes a target
molecule within the meaning of embodiments of the invention. a
hybridization of nucleic acid sequences.
According to embodiments, the fluorescence-inducing molecules are
fluorophores (e.g. an antibody labeled with a fluorophore).
According to alternative embodiments, the fluorescence inducing
molecules are enzymes that trigger the emission of fluorescence
signals by other molecules in spatial proximity to the
fluorescence-inducing molecules (e.g. the horse radish
peroxidase--HRP--enzyme: alone, the HRP enzyme, or conjugates
thereof, is invisible; its presence must be made visible using a
substrate that, when oxidized by HRP using hydrogen peroxide as the
oxidizing agent, yields fluorescent signal).
Said features may be advantageous, as a plurality of commercially
available kits exist for labeling different biomarkers with
different fluorescent dyes. For example, the use of
primary-secondary antibody labeling systems allows to use well
established staining protocols for a particular fluorescent dyes
for a large variety of different biomarkers simply by using
different primary antibodies which all act as specific binding
partner of a secondary antibody coupled to a fluorophore.
According to embodiments, the fluoro dot comprises a known density
of further fluorescence-inducing molecules of at least one further
type of fluorescence-inducing molecules. The tissue sample and the
target dot both have been stained in addition with the further
fluorescence-inducing molecules. The method comprises: receiving a
further digital image of the slide, the further digital image
comprising intensity values of the tissue sample, intensity values
of the target dot and intensity values of the fluoro dot, the
intensity values of the fluoro dot, the target dot and the tissue
sample correlating with the number of the further
fluorescence-inducing molecules in the fluoro dot, the target dot
and the tissue dot, the further fluorescence-inducing molecules
being adapted to directly or indirectly bind to the target
molecules during the staining procedure; receiving: further pixel
intensities of the fluoro dot depicted in the further image;
further pixel intensities of the target dot depicted in the further
image; further pixel intensities of the area of the tissue sample
depicted in the further image; further fluorescence-inducing
molecule density information indicative of the known density of the
further fluorescence-inducing molecules in the fluoro dot;
computing a further density of target molecules in the area of the
tissue sample as a function of the further pixel intensities of the
fluoro dot, the target dot, the area of the tissue sample, the
further density information of the further fluorescence-inducing
molecules and the target molecule density information.
For example, the receiving of the intensity information may be
implemented as follows: the light source may emit light of a
different excitation wavelength that selectively induces a
fluorescence signal of the further fluorescence-inducing molecule,
not of the first fluorescence-inducing molecule. Thereby, two
different digital images are obtained which are analyzed as before
for determining the respective target molecule densities from the
one of the digital images whose pixel intensities are generated by
florescence signals emitted by a respective one of the first or
further fluorescence-inducing molecule.
Alternatively, a multispectral light source may be used, but
different fluorescence filters may be used; for example, a first
fluorescence filter may be used by the image acquisition system to
receive a first digital image that selectively comprises
fluorescence signals of the first fluorescence-inducing molecules
(of the whole slide having been subjected to a staining procedure
using the first fluorescent-inducing molecule, the first image
including fluorescence signals of the fluoro dot, the target dot
and of the tissue sample emitted by the first fluorescence-inducing
molecules); a second fluorescence filter may be used by the image
acquisition system to receive a second digital image that
selectively comprises fluorescence signals of the further
("second") fluorescence-inducing molecules (of the whole slide
having been subjected to the same or a different staining procedure
using (also) the second fluorescence-inducing molecules, the second
digital image including fluorescence signals of the fluoro dot, the
target dot and of the tissue sample emitted by the further
fluorescence-inducing molecules).
Depending on the staining protocols and the primary antibodies
used, the two images may be used for quantifying the same type of
target molecule twice (e.g. for obtaining a more accurate
estimation of the target molecule density which is based on the use
of two independent staining approaches with two different
fluorescent labels, or for quantifying multiple different target
molecules in the same tissue concurrently.
Like the density information of the first fluorescence-inducing
molecules, the further fluorescence-inducing molecule density
information indicative of the known density of the further
fluorescence-inducing molecules in the fluoro dot can be received
by the image analysis system e.g. by reading respective data values
from a storage medium, e.g. a non-volatile storage medium.
Thus, multiple biomarkers may be stained with different
fluorophores often within the same staining protocol.
Said features may be advantageous, because a slide whose fluoro dot
comprises two or more types of fluorescence-inducing molecules may
allow the accurate determination of the densities of multiple
different target molecule types provided said different target
molecule types are labeled with a fluorescence-inducing molecule
that is comprised in the fluoro dot.
Alternatively, the same type of target molecules in the target dot
and in the tissue sample of the slide may be stained with different
fluorescence-inducing molecules.
According to embodiments, the method further comprises computing
the average of: the density of the target molecules in the area of
the tissue sample computed according to any one of the embodiments
described herein, and the further density of the target molecules
in the area of the tissue sample computed according to any one of
the embodiments described herein.
Thus, by performing the intensity calibration with the two
calibration dots for each of the two or more fluorescent-inducing
molecule types separately, and then e.g. computing an average
target molecule density value, an even more accurate determination
of the number of target molecules in a particular tissue sample may
be achieved.
According to embodiments, the target molecule is selected from a
group comprising: a biomarker molecule; a primary antibody capable
of selectively binding to the biomarker molecule in a predefined
molecular ratio; a secondary antibody capable of selectively
binding to the primary antibody in a predefined molecular
ratio.
In a further aspect, the invention relates to an image analysis
system configured for determining the density of target molecules
in a tissue sample depicted in a fluorescence image. The system
comprises a storage medium and one or more processors configured
for: receiving a digital image of a slide, the slide comprising the
tissue sample, a target dot and a fluoro dot, the fluoro dot
comprising a known density of fluorescence-inducing molecules, the
target dot comprising a known density of target molecules, the
tissue sample and the target dot having been stained in the same
staining procedure with the same type of fluorescence-inducing
molecules as contained in the fluoro dot, the digital image
comprising intensity values of the tissue sample, intensity values
of the target dot and intensity values (120) of the fluoro dot, the
fluorescence-inducing molecules being adapted to directly or
indirectly bind to the target molecules during the staining
procedure; receiving: pixel intensities of the fluoro dot depicted
in the image; pixel intensities of the target dot depicted in the
image; pixel intensities of an area of the tissue sample depicted
in the image; fluorescence-inducing molecule density information
indicative of the known density of the fluorescence-inducing
molecules in the fluoro dot; target molecule density information
indicative of the known density of the target molecules in the
target dot; computing the density of target molecules in the area
(154) of the tissue sample as a function of the pixel intensities
of the fluoro dot, the target dot, the area of the tissue sample,
the fluorescence-inducing molecule density information and the
target molecule density information.
In a further aspect, the invention relates to a slide comprising a
tissue sample, a target dot and a fluoro dot, the fluoro dot
comprising a known density of fluorescence-inducing molecules, the
target dot comprising a known density of target molecules.
According to embodiments, the slide with the tissue sample and the
target dot has been stained in the same staining procedure with the
same type of fluorescence-inducing molecule as contained in the
fluoro dot. The fluorescence-inducing molecules is adapted to
directly or indirectly bind to the target molecules (known to be
contained in the target dot and suspected of being contained in the
cells of the tissue sample) during the staining procedure.
According to embodiments, the fluoro dot of the slide comprises a
known density of further fluorescence-inducing molecules of at
least one further type of fluorescence-inducing molecules. The
further fluorescence-inducing molecules are adapted to directly or
indirectly bind to the target molecules during the staining
procedure.
This type of slides may be used for computing the target molecule
density in the tissue sample as an average of the target molecule
densities obtained individually for the different
fluorescence-inducing molecules. "Different" fluorescence inducing
molecules as used herein are fluorescence-inducing molecules having
a distinguishable fluorescence emission spectrum.
According to other embodiments, the fluoro dot of the slide
comprises a known density of further fluorescence-inducing
molecules of at least one further type of fluorescence-inducing
molecules. The further fluorescence-inducing molecules are adapted
to directly or indirectly bind to further target molecules of a
further target molecule type during the staining procedure. The
target dot in addition comprises a known density of the further
target molecules. For example, the first and the further ("second")
target molecules can be two different biomarkers.
According to embodiments, the target dot is an area of the slide
that is coated with a known number of the target molecules or that
is coated with a known number of cells, the cells expressing or
comprising a known number of the target molecules.
An "image analysis system" as used herein is an electronic system,
e.g. a computer, configured for extracting meaningful information
from digital images by means of digital image processing
techniques. Image analysis tasks can comprise color deconvolution,
connected component analysis, image segmentation and/or edge
detection for identifying dots, tissue samples, individual cells,
the type of the cells (tumor or stroma cell, different types of
immune cells) and the like. In some embodiments, an image analysis
system can further comprise or be operatively coupled to an image
acquisition system, e.g. a microscope.
A "target molecule" as used herein is a molecule, molecule part
(e.g. a gene sequence contained in genomic DNA), or molecule
complex (e.g. an epitope bound to one or more intermediary
molecules, the intermediary molecule(s) being the actual binding
partner(s) for a fluorescence-inducing molecule) whose quantity in
a tissue sample shall be determined. For example, the target
molecule can be a biomarker, or biological marker, i.e., an
indicator of some biological state or condition. Biomarkers are
often measured and evaluated to examine normal biological
processes, pathogenic processes, or pharmacologic responses to a
therapeutic intervention. A known density of the target molecule is
attached to a region of the slide which is referred herein as
"target dot". A target molecule as used herein can also be a
specific sub-sequence of a nucleic acid molecule that is of
particular interest, e.g. a gene sequence in a DNA molecule.
A "fluorescence-inducing molecule" as used herein is a molecule
that is itself capable of emitting fluorescent light in response to
absorbing light or other electromagnetic radiation (a
"fluorophore", e.g. an antibody coupled to fluorescein) or is a
molecule that is capable of inducing fluorescence in specific other
molecules in its spatial neighborhood (e.g. a HRP complex). In most
cases, the emitted light has a longer wavelength, and therefore
lower energy, than the absorbed radiation. Many fluorescent stains
have been designed for a range of biological molecules. Some of
these are small molecules which are intrinsically fluorescent and
bind a biological molecule of interest. Major examples of these are
nucleic acid stains like DAPI and Hoechst (excited by UV wavelength
light) and DRAQ5 and DRAQ7 (optimally excited by red light) which
all bind the minor groove of DNA, thus labeling the nuclei of
cells. Others are drugs or toxins which bind specific cellular
structures and have been derivatised with a fluorescent reporter. A
major example of this class of fluorescent stain is phalloidin
which is used to stain actin fibres in mammalian cells. There are
many fluorescent molecules called fluorophores or fluorochromes
such as fluorescein, Alexa Fluors or DyLight 488, which can be
chemically linked to a different molecule which directly or
indirectly binds to a target molecule of interest within the sample
and within the target dot.
A "fluorescence microscope" as used herein is an optical microscope
that uses fluorescence and phosphorescence instead of, or in
addition to, reflection and absorption to study properties of
organic or inorganic substances. A "fluorescence microscope" refers
to any microscope that uses fluorescence to generate an image, also
referred to as "digital fluorescent image", whether it is a more
simple set up like an epifluorescence microscope, or a more
complicated design such as a confocal microscope, which uses
optical sectioning to get better resolution of the fluorescent
image.
A "tissue sample" as used herein is matter having been gathered
from the body of an organism, e.g. a mammal, e.g. a human. The
tissue sample can be, for example, a tumor tissue sample, a blood
smear sample, a skin tissue sample, or the like. Tissue samples can
be stained with fluorescent or other stains and can be attached to
microscope slides for enabling an image acquisition system to
generate digital images of the sample that can be analyzed. The
image analysis can be performed e.g. to aid in the process of a
medical diagnosis and/or evaluation of an indication for treatment,
further medical tests or other procedures.
A "fluorescence image" as used herein is a digital image whose
pixels have pixel intensities which are indicative of fluorescence
signals captured by a fluorescence microscope or by another
fluorescence-based image acquisition system.
A "dot" as used herein is an area of a slide. The dot may have any
shape, e.g. a rectangular, square or circular shape.
The "density" of a molecule in a dot as used herein is the number
of molecules in a given area unit, e.g. mm.sup.2. A "known"
molecule density means that the density of the respective molecule
type was already known when an image analysis method for
quantifying target molecules in a tissue sample is carried out. For
example, the molecule density can have been determined empirically
in advance before or while a particular calibration dot was created
on the slide.
A "slide" as used herein is a carrier structure for tissue samples
that is used for generating digital images of the tissue sample, in
particular fluorescence images. Preferentially, the slide is a
microscope slide. The slice can be e.g. a glass slide that can be
coated with one or more layers that facilitate or enable the
adhesion of target molecules, fluorescence-inducing molecules
and/or reference cells to the slide.
A "tyramide signal amplification staining protocol", also called
"TSA assay" or "CARD" for Catalyzed Reporter Deposition, is an
enzyme-mediated detection method that utilizes the catalytic
activity of an enzyme (typically horseradish peroxidase--HRP) to
generate high-density labeling of a target protein or nucleic acid
sequence in situ. TSA has been reported to increase detection
sensitivity up to 100-fold, as compared with conventional
avidin-biotinylated enzyme complex (ABC) procedures. Moreover, for
multiparameter detection of targets in either live or fixed cells
or tissues, TSA can be combined with several other important
technologies, including our nucleic acid labeling kits, primary and
secondary antibodies, avidin and lectin conjugates, cytoskeletal
stains, and others. According to preferred embodiments, TSA
labeling is a combination of at least three elementary processes
that typically comprise: a) binding of a probe to the target via
immunoaffinity (proteins) or hybridization (nucleic acids) followed
by secondary detection of the probe with an HRP-labeled antibody or
streptavidin conjugate. Peroxidase conjugates of other targeting
proteins such as lectins and receptor ligands are likely to be
suitable for labeling targets, as is endogenous peroxidase
activity; b) activation of multiple copies of a labeled tyramide
derivative by HRP; often, a fluorescent or biotinylated tyramide is
used; c) covalent coupling of the resulting highly reactive,
short-lived tyramide radicals to residues (principally the phenol
moiety of protein tyrosine residues) in the vicinity of the
HRP-target interaction site, resulting in minimal diffusion-related
loss of signal localization. Using a slide with a fluoro dot and a
target dot in the context of a TSA assay may be particularly
advantageous because the increased signal strength of the TSA assay
is typically accompanied by a high variability of the signal
amplification strength and thus with a large uncertainty regarding
the actual amount of target contained in a tissue sample. Using a
slide with a fluoro dot and a target dot in the context of a TSA
assay may allow benefitting from the high sensitivity of the TSA
protocol and at the same time being able to estimate the amount of
target molecules in the sample.
BRIEF DESCRIPTION OF THE DRAWINGS
In the following embodiments of the invention are explained in
greater detail, by way of example only, making reference to the
drawings in which:
FIG. 1 is a block diagram of a tissue slide;
FIG. 2 is a block diagram of an image analysis system configured
for analyzing a fluorescence image of a slide;
FIG. 3 is a flow chart of a fluorescence image analysis method for
quantifying target molecules in a tissue sample;
FIG. 4 depicts three different tyramide signal amplification
assays;
FIG. 5 is a fluorescence image of a slide according to embodiments
of the invention.
DETAILED DESCRIPTION
FIG. 1 shows a slide 150 that comprises a fluoro dot FD, a target
dot TD and a tissue sample. The slide can be, for example, a glass
tissue slide. In a first area FD referred herein as "fluoro dot",
the slide is covered by fluorescence-inducing molecules of a known
molecule density. In a second area TD referred herein as "target
dot", the slide is covered by target molecules of a known molecule
density. In a third area of the slide, the slide comprises a tissue
sample 152, e.g. of a mammal, e.g. a tissue sample of tumor tissue
of a patient.
Typically, the slide is used for examining the location of a
particular type of molecule of interest, referred herein as "target
molecule" and for determining the density (e.g. the "expression
level" in case the target molecule is a protein) of the target
molecules in the tissue sample. For example, the target molecules
can be a biomarker for a particular tumor-subtype, e.g. Her2,
BRCA1, BRCA2 or the like.
The target dot TD comprises a known density of the target molecule.
Several different methods can be used for generating a target dot
having a known target molecule density. For example, the slides can
be coated with a defined number of isolated target molecules or
with a defined number of reference cells whose target molecule
concentration is known. For example, the target molecule
concentration and the reference cells can be determined by
homogenization of a reference tissue known to comprise the target
molecules, generating a suspension of the reference cells,
empirically determining the amount of target protein in the
reference cells, e.g. by mass spectrometry or by fluorescence
cytometry or other appropriate technical means. Then, either a
defined number of reference cells is attached to the surface of the
slide 150, or the suspension of reference cells is used for
attaching an unknown number of reference cells on the surface of
the slide and later counting the number of reference cells per mm2
which have been successfully attached to the surface of the slide.
This counting can be performed manually or automatically by means
of image analysis techniques which are able to automatically
identify and count cells. The same homogeneous suspension of
reference cells can be used for coating a plurality of slides, e.g.
several hundred, several thousand or even more slides. As a
homogeneous suspension of reference cells was used for coating the
tissue slide, the determination of the reference cell density in
the target dots of only a few slides may be sufficient for
determining and "knowing" also the reference cell density of the
target dots of all the other slides coated based on the same
homogeneous reference cell suspension. As the average target
molecule concentration in each reference cell was also determined
empirically in the reference cells, it is possible to determine and
compute the target molecule density in the target dot TD. This
"known" target molecule density can optionally be printed onto the
slide 150 and/or can be stored in a storage medium.
The fluoro dot FD comprises a known density of the
fluorescence-inducing molecules molecule. Several different methods
can be used for generating a fluoro dot having a known
fluorescence-inducing molecule density. For example, the slides can
be coated with a defined number of isolated fluorescence-inducing
molecules. Alternatively, a large number of slides can be coated
with the same homogeneous suspension (or solution) of
fluorescence-inducing molecules. The density of the
fluorescence-inducing molecules that actually become firmly
attached to the slides to form the fluoro dots can later be
empirically determined, e.g. by means of mass spectrometry or other
appropriate analytical methods. Provided, the same
suspension/solution of fluorescence-inducing molecules was used for
coating the slides, it is sufficient to empirically determine the
reciting fluorescence-inducing molecule density on only a few
slides and respective fluoro dots. This "known"
fluorescence-inducing molecule density can optionally be printed
onto the slide 150 and/or can be stored in a storage medium.
Preferably, the fluoro dot actually comprises a mixture of two or
more different types of fluorescence-inducing molecules, whereby
the density of each of that different types of
fluorescence-inducing molecules is known (the densities of said
different types of molecules may or may not be identical). This may
allow a user to flexibly choose a particular fluorescent dye for
staining a target molecule of interest among a plurality of
different fluorescent dyes which are commercially available. Thus,
a user is not limited to a single a particular fluorescent dye or
fluorescence-inducing molecule that was used for generating the
fluoro dot. Moreover, a user of a slide comprising a fluoro dot
with two or more different fluorescence-inducing molecule types can
perform multi-color fluorescence image analysis and can stain and
quantify multiple different types of target molecules in a single
experimental procedure. For example, the user can use different
fluorescence filters or can use a light source with a defined
excitation spectrum to induce the emission of fluorescence signals
selectively by a particular one of the fluorescence-inducing
molecules in the fluoro dot (and the target dot and the tissue
sample of the slide), thereby creating a fluorescent image that
selectively indicates the presence of this particular
fluorescence-inducing molecule (and any target protein to which it
is selectively attached).
The whole slide with the target dot, the fluoro dot and the tissue
sample is subjected to a staining procedure during which the same
fluorescence-inducing molecule that is contained in the fluoro dot
becomes selectively attached to the target molecules in the target
start and to the target molecules in the tissue sample 152, if any.
Although the actual number of fluorescence-inducing molecules which
become attached to a single target molecule may be unknown, e.g.
because that attachment ratio strongly depend on the
particularities of the staining procedure, it can safely be assumed
that the attachment ratio is the same for the target molecules in
the target dot as for the target molecules in the tissue sample
152. As the target molecule density in the target dot and the
fluorescence-inducing molecules in the fluoro dot are known and as
the fluorescence signal intensities of the target starts, the
fluoro dot and the tissue sample 152 have been captured by the same
image acquisition system (e.g. the same fluorescence microscope),
the target dot and the fluoro dot can be used for calibrating the
intensity information obtained for the tissue sample and can be
used for accurately determining the target molecule density in the
tissue sample 152.
In order to generate the target dot of one or more slides as
depicted in FIG. 1, a reference tissue is taken from a tissue known
or suspected to comprise a particular target molecule. For example,
a tissue biopsy can be prepared as formalin-fixed paraffin-embedded
(FFPE) sample. FFPE preps can be stored indefinitely at room
temperature, allowing the nucleic acids (both DNA and RNA) to be
recovered and analyzed even decades later.
In order to generate the target dot, a slice or punch from an FFPE
reference sample is de-paraffinized and a suspension of reference
cells stained for one or more types of target molecules (e.g.
keratin, vimentin or a particular DNA/RNA sequence) is generated
from the reference sample. Flow cytometry is then applied to all or
a subset of the reference cell in the suspension to determine the
number of target molecules in the reference cells. Optionally, the
reference cells in the suspension can be sorted to remove cells
which do not comprise the target protein or which do not comprise
the target protein in a desired quantity range. For example,
commercially available systems and methods such as the DEPArray
system's image-based sorting can be used for selectively obtaining
reference cells which express the target protein in a desired
amount range. Thus, a highly pure collection of reference cells
with known target molecule density can be recovered from FFPE
tissue samples and can be used for coating a specific area of the
tissue slide with the reference cells. The density of reference
cells that actually become firmly attached to the target dot may be
empirically known from previous coating procedures under the same
coating conditions or may be determined for one or more of the
generated target dots later by manually or automatically counting
the cells. Thus, the target molecule density in the target dot is
computed (and thus "known") from the empirically determined target
molecule concentration in the reference cells and from the density
of reference cells which become firmly attached to the slide,
thereby forming the target dot. The creation of homogeneous cell
blocks and the attachment of said cell blocks on slides, e.g. for
the purpose of providing a positive control for biomarkers in
immunohistochemistry experiments, is described for example in U.S.
Pat. No. 9,395,283 B1 which is incorporated herewith by reference
in its entirety.
For example, reference cells known to comprise a particular target
molecule of interest are pre-treated and stained with a fluorescent
dye or other fluorescence-inducing molecule. Then, the target
molecule density in the cell block is empirically determined. The
density of the reference cells within the final block is controlled
by adjusting the size of a mold used for forming the cell blocks in
order to produce a target dot comprising cells of a certain
number/density/count of target molecules, wherein the target dot
comprises a particular number of reference cells. The reference
cell blocks have a defined size and length to control the number of
reference cells in each block, thereby producing a target dot
comprising a certain number (i.e. pre-designated, "known" number)
of reference cells with a known density (or concentration) of
respective target molecules.
The steps in the method of making the target dot (i.e. cell block)
comprise: a) passing reference cells through a cell collection
device, wherein said reference cells are in suspension, fixed
pellet, or unfixed pellet form; b) performing reference cell
counting; c) fixing the cells in a composition comprising
paraformaldehyde in PBS to create a "cell pellet" or "cell block";
d) preparing the molds; re-suspending the cells in PBS; and
immobilizing the suspension in controlled temperature; e) injecting
the cell suspension into the prepared mold of predefined size and
shape at a controlled temperature; f) cooling and treating the
cells with a paraffin processor; and, g) performing molecule
quantification, e.g. DNA extraction and quantification or mass
spectrometry; the information obtained during this step may in
addition be used to check and verify that the reference cells are
distributed homogeneously in the cell block.
The cell block preparation step in the above described method of
making the cell block comprises: a) passing reference cells through
an apparatus to create a homogenous mixture of immobilized cells;
b) injecting the homogeneous reference cell mixture into a Mold A
("first mold", e.g. a mold being 4 mm in radius, 145 mm in length),
and remove the generated reference cell blocks from the mold; and,
c) processing cell blocks with paraffin, removing individual blocks
from paraffin, and embedding the processed cell blocks into a Mold
B ("second mold", e.g. a cubed paraffin Mold of 2 cm *2 cm *2 cm).
The cell blocks are wrapped with parafilm and kept in air-tight box
at 4.degree. C. until they are sectioned in multiple tissue block
sections that are attached to the slide and are respectively used
as target dots.
According to embodiments, reference cell blocks of a specific,
known reference cell density are created by mixing the suspension
of reference cells with agarose, e.g. 3% agarose, whereby the
amount of agarose is chosen such that the resulting reference
cell/agarose mixture that is to be filled into the second mould has
the desired reference cell density.
Preferentially, the reference cells attached to the slide are
homogeneously distributed in the target dot. The detection of
homogeneity can be performed using cell counting, e.g. by digital
immunohistochemistry devices (e.g. Aperio ScanoScope). Detection of
homogeneity can also be confirmed by the extraction and
quantification of nucleic acids from each cell block section to
determine the amount of nucleic acids in each block and the ratio
of a mixture of cells within the block. Methods of DNA extraction
and quantification are, for example, PCR, digital PCR and/or
sequencing methods.
Alternative methods of creating the target dot can be used as well:
for example, in case the target molecule is a protein that is
available or can be prepared in purified form, protein coating
techniques as used for the production of protein microarrays can be
used for firmly attaching the protein in a desired amount on the
surface of the slide. Several techniques of attaching proteins to
slide surfaces are disclosed e.g. in the paper "Systematic
comparison of surface coatings for protein microarrays" of Birgit
Guilleaume et al., Proteomics 2005, 5, 4705-4712 4705, WILEY-VCH
Verlag GmbH & Co. KGaA, which is incorporated herewith by
reference in its entirety. Further documents which describe a
method of coating of modified glass slides with a particular
protein which can be applied for generating the target are, for
example: Peluso, P., Wilson, D. S., Do, D., Tran, H. et al., Anal.
Biochem. 2003, 312, 113-124. Zhu, H., Klemic, J. F., Chang, S.,
Bertone, P. et al., Nature, Genet. 2000, 26, 283-289. Mac Beath,
G., Schreiber, S. L., Science 2000, 289, 1760-1763. Stillman, B.
A., Tonkinson, J. L., Biotechniques 2000, 29. 630-635.).
The above cited documents mainly involve covalent attachment of
proteins via different functional groups that are coupled to the
surface. The most commonly used functional groups are epoxy,
aldehyde, and Nhydroxysuccinimide (NHS)-ester. In contrast, am
inosilane coatings immobilize proteins via electrostatic
forces.
According to some embodiments of the method, the method comprises
generating the fluoro dot by coating an area of the slide with
fluorescence-inducing molecules.
For example, US patent US2003/0015668, which is incorporated in
this patent specification by reference in its entirety, discloses a
method to deposit an extremely thin layer of Cy3, Cy5 or other
fluorescent dye doped glass by evaporation or sol-gel process on a
non-fluorescent support.
According to another example, the fluoro dot can be created by any
one of the calibration dot creation methods descried in EP 1774292
B1 which is incorporated in this patent specification by reference
in its entirety. Said document describes rare-earth ion doped
inorganic arrays for calibration of fluorescence microarray
scanners and a process of making them. Surfactants or dispersants
are employed to help the coated inorganic phosphors disperse evenly
in aqueous suspensions. Any suitable one or mixture of the
surfactants, e.g., Tween-20, Triton-100, sodium lauryl sulfate
(SLS), polyethylene glycol (PEG) 2000, PEG 4000, PEG 6000, PEG
8000, PEG 10000, PEG 20000, polyvinyl alcohol (PVA), polyethylene
imine (PEI), sodium polyacrylate (PAA) can be used in the
suspension of the inorganic phosphors. The quantity of said
surfactants may be 0.1%.about.10%, more preferably 0.1-5%,
Preferably, a spotting agent, e.g., dimethyl sulfoxide (DMSO) or
glycerol is added in said suspension of inorganic phosphors. The
slide may be prepared by the following methods: the fluoro dot is
spotted on the slide by a contact printer or by spin coating and
screen printing techniques. The diameter of the fluoro spots can
e.g. be in a range of 100-500 .mu.m, more preferably 120-300 .mu.m,
but it can also have any other size.
The fluoro dot and the target dot can be applied on a standard
microscope glass slide having e.g. a dimension of 75.6 mm.times.25
mm.times.1 mm. The surface of the glass slide may be unmodified or
modified. For example, the glass slide to carry the fluoro dot can
be globally or locally modified by a chemical method, i.e.
amino-modified slide, aldehyde-modified slide, epoxy-modified
slide, thiol-modified slide or polymer film modified slide, i.e,
PVA film, agarose film, or the mixing of PVA and agarose film. A
very thin layer can be deposited on the slide surface to protect
the fluoro dot comprising said inorganic phosphors, Thus, a
transparent thin film of polydimethylsiloxane (PDMS) or PVA with
low fluorescence background may be created, whereby the thickness
of the film is preferably less than 50 .mu.m. Further details of
the method are described in EP 1774292 B1, In dependence on the
fluorescence-inducing molecule used, the protocol may be adapted to
the particular property of the respective fluorescence-inducing
molecule.
FIG. 2 is a block diagram of an image analysis system 100 according
to an embodiment of the invention. The system comprises one or more
processors 104, a main memory 106 and a non-volatile storage medium
108. The storage medium comprises computer-interpretable
instructions 110, 126 configured for performing an image analysis
method for determining the density of the target molecules in a
tissue sample 152 as described herein for embodiments of the
invention and as depicted in the flowchart of FIG. 3. The system
may optionally comprise or be coupled to an image acquisition
system 124, e.g. a camera, for receiving a fluorescence image 130
of a slide 150 as depicted, for example, in FIG. 1. In addition, or
alternatively, the fluorescence image 130 is stored on the storage
medium 108. The storage medium 108 may comprise instructions for a
dot detection log 126 which is configured for performing an image
analysis of a whole slide image 130 of the fluorescence slide for
automatically detecting the regions in the digital image 130 which
corresponds to the fluoro dot, the target dot and the tissue sample
152. In addition, or alternatively, the coordinates of the two
calibration dots can be pre-configured and stored in the storage
medium and can be used for quickly and accurately identifying the
pixels in the digital image which corresponds to the respective
calibration dots.
Thus, in step 202 the image analysis system 100 may retrieve a
digital fluorescence image 130 of the slide 150 directly via the
corner out 124 or by reading an existing fluorescence image 130
from the storage medium 108.
Then in step 204, pixel intensities of different regions of the
image which corresponds to the target dot, the fluoro dot or the
tissue sample are received. For example, a dot detection logic 126
automatically identifies pixel regions corresponding to the fluoro
dot, to the target starts and to the tissue sample. Then, the image
analysis logic 110 can compute the average intensity 122 of the
pixels corresponding to the target dot, the average intensity 120
of the pixels corresponding to the fluoro dot and the average
intensity 118 of an area of interest 154 within the tissue sample.
The area of interest 154 can be a single pixel (whose intensity
value is used instead of the "average" intensity value) or can be a
set of pixels whose combined analysis is still considered as
sufficiently detailed to extract the biomedical information of
interest. The number of pixels that constitutes the area of
interest 154 may depend on the particular experimental setting and
the medical question, but typically comprises fewer pixels than are
contained in a pixel area typically corresponding to the size of a
cell. Preferably, the fluorescence intensity values of all pixels
corresponding to the tissue sample are analyzed and calibrated such
that the real number of target molecules in the respective tissue
sample region can be determined. Thus, the determination of target
molecule density region 154 is described just as an example for
demonstrating how the totality of tissue sample pixels can be
analyzed according to embodiments of the invention. The obtained
intensity information 122, 120 and 118 can optionally be stored in
the storage medium 108.
Then, the image analysis logic 110 reads the known density
information 116 indicating the target molecule density in the
target dot TD and the known density information 114 indicating the
fluorescence-inducing molecule density in the fluoro dot FD.
Then in step 206, the image analysis method 110 computes the target
molecule density in the area 154 of interest as a function of the
measured intensity values 1 to 2, 120 and 118 and the "known"
molecule densities 114, 116 of the respective calibration dots TD,
FD.
FIG. 4 depicts three different tyramide signal amplification assays
for which respective kits are commercially available. Tyramide
Signal Amplification (see e.g. U.S. Pat. Nos. 5,731,158, 5,583,001
and 5,196,306) amplifies fluorescent signals in many applications.
TSA can be used for increasing sensitivity in any application that
allows the addition of horseradish peroxidase (HRP) into its
protocol, such as immunohistochemistry, in situ hybridization,
ELISA and microarray-based differential gene and protein expression
studies. HRP is used to catalyze the deposition and binding of a
labeled (e.g., biotin, DNP or other labeling moieties) tyramide
onto tissue sections or cell preparations comprising a target
molecule, typically a biomarker, e.g. a protein such as CD20, CD4,
CD45RO, CD68, FOXP3, panCK, and other custom targets. The binding
is covalent because the reaction intermediately dimerizes with
tyrosine residues on the surface-bound endogenous proteins. These
labels can then be detected by standard fluorescent techniques.
Since the added labels are only deposited proximal to the enzyme
site, the fluorescence signal is selectively indicative of the
presence and amount of a target molecule. If the fluoro dot
comprises fluorescence-inducing molecules of two or more different
types and if the slide 150 is labeled with two or more respective
fluorescence-inducing molecules, multi-target detection in the same
sample is of interest is supported. A further beneficial aspect of
using TSA is that unamplified detection levels can be maintained
while utilizing up to 1,000-fold less primary antibody.
The TSA amplification reagent is a phenolic compound that, when
activated by HRP, is converted into a short-lived, extremely
reactive free radical intermediate. This free radical intermediate
reacts rapidly with and covalently binds to electron-rich regions
of adjacent proteins (predominantly tyrosine residues). This
binding occurs adjacently to the sites at which the HRP enzyme is
bound. HRP catalyzes the deposition and covalent binding of the
fluorophore- or hapten-labeled TSA substrate onto tissue sections
comprising the target molecules. In FIG. 4, an antigen (Ag 402) may
represent an epitope of a target molecule of interest. The target
molecule may be attached e.g. via an adhesive layer 410 directly to
the slide 150, e.g. in the target dot TD, or can be attached to the
slide as an element a cell (e.g. a reference cell in the target dot
or a tissue cell in the tissue sample). The black antibody 404
represents a primary antibody that selectively binds to the
antigen. The grey antibody 406 is a secondary antibody that
together with the HRP enzyme forms a secondary antibody/HRP
conjugate. "T" is a tyramide with fluorescent label for
visualization. The fluorophore-labeled TSA substrate can be, for
example, a biotin-tyramide conjugate, cyanine-3-tyramide conjugate
or a DNP (dinitrophenol)-tyramide conjugate.
By increasing the sensitivity 10-200 times that of standard
ICC/IHC/ISH methods, applying a TSA assay on a slide comprising a
target dot and a fluoro dot and applying the image analysis method
as described herein for embodiments of the invention allows to
accurately quantify even low-abundance targets.
FIG. 5 shows a fluorescence image 502 of a slide according to
embodiments of the invention. Pixel region 504 corresponds to the
target dot TD, pixel region 506 corresponds to the fluoro dot FD
and tissue sample pixel region 508 corresponds to a tissue sample
152. For example, the pixel regions 504, 506 and 508 can be
automatically identified by various image analysis techniques such
as blob detection, image segmentation, and/or edge detection
methods. By changing the fluorescence filter or the excitation
light source and the respective excitation wavelength, a further
fluorescence image can be generated whose intensities are
indicative of a different target protein labeled with a different
fluorescence-inducing molecule or whose intensities are indicative
of the same target protein labeled with the different
fluorescence-inducing molecule.
* * * * *